Abstract: Accurate quantitative measurement is a difficult problem in protein and antibody purification. Traditional methods like Bradford or BCA are very complicated and take a long time. It's very rapid and convenient to quantitatively measure protein UV absorbance.
Keywords: Protein UV Absorbance, UV Absorption of Protein, Protein Concentration Measurement
1. Calculating Protein Concentration from Absorbance
The UV absorption of protein comes from aromatic amino acids, e.g. tryptophan and tyrosine molecules contain conjugated double bond. The light absorption peaks near 280 nm wavelength. Measurement of protein absorbance at 280 nm wavelength is the most simple and convenient technique for analyzing protein content in the solution, because tyrosine and tryptophan residues exist in most proteins.
2. Methods to Determine Protein Concentration
The great advantage of this technique is rapid and samples are not damaged. However, the measurement is based on the strong absorption value of tyrosine(Tyr), phenylalanine(Phe), tryptophan(Trp) residues. The protein content varies accordingly. The quantitation is inaccurate if the protein is absence of these amino acids. Moreover, nucleic acid and other substances containing conjugated pi bonds attached to a benzene ring (e.g. imidazole for protein elution) can influence the quantitation also. These things should be dialysed before the detection.
3. Protein Concentration Measurement by Microspectrophotometer
Step 1: Open the software and select Protein A280;
Step 2: Select sample type in the top right-hand corner. Option for measuring unknown proteins: 1Abs=1mg/ml(Select the default parameter IgG for antibody concentration detection. Devices of most manufacturers have this parameter.);
Step 3: Wash cuvette with pure water three times. Select suitable solution as the background. Drop 2 µ L and click “blank” to remove the background. Repeat the operation again and click “measure”. The A280 value of the sample displayed below is the concentration;
Step 4: Visit expasy to search extinction coefficient by entering protein sequence. The final concentration is obtained by using the measured A280 to divide the extinction coefficient.
4. Validated FineTest Protein
FineTest strictly controls antibody and protein product quality. QC strategies include SDS-Page analysis, concentration comparison between lyophilized standard and BSA protein, concentration and purification validation and correction by referring to the data of imported microspectrophotometer etc.
Step 1: 12% SDS-PAGE Validation (1mg BSA standard, loading amount: 10ul);
Step 2: Analyse protein sequence and calculate extinction coefficient;
Step 3: Concentration determination result offered by microspectrophotometer.
